1. Field of the Invention
The present invention relates to antibodies to human gastrin-releasing peptide (GRP) precursor, and the use of the antibodies as a diagnostic agent for cancer.
2. Related Art
It is known that cancer cells produce substances specific to the cancer cells as well as substances common between normal cells and cancer cells. It has become possible to diagnose the characteristics of cancer cells and patients with cancer by measuring the cancer-specific substance. Substances specifically produced by cancer cells include oncogene products and growth factors, which are responsible for oncogenesis, growth and developments of cells. Moreover, it is considered that the production of carcinoembryotic proteins, hormones and enzymes and the like are characteristics of the oncogenesis. Therefore, if one of substances which define cancer cells, i.e., so-called tumor markers can be assayed with high sensitivity, diagnosis of cancers becomes possible.
Since the presence of neuroendocrine particles in small cell lung cancer was observed in 1968, it has been asserted that small cell lung cancer is derived from neuroendocrine cells, and at present, is included in APUD (amine precursor uptake and decarboxylation)-type tumors, and is distinguished from other non-small cell lung cancers which are epithelial tumors. It has been known that the small cell lung cancer shows the characteristics of the neuroendocrine cells in a cytobiological study, and is reported to produce peptide hormones such as serotonin, adrenocorticotropic hormone (ACTH), calcitonin, gastrin releasing peptide (GRP) etc., to exhibit high L-dopa decarboxylase (L-DDC) activity characteristic to APUD-type cells or tumors, and to exhibit high activities of neuron specific enolase (NSE) and creatine kinase BB (CK-BB), which are specific to neuron cells.
Analysis of a surface antigen of small cell lung cancer based on various biological properties of the cells as indicators using a monoclonal antibody was started with a report by Minna, Science 214, 1246-1248, 1981, and has been well developed by a lot of researchers. Assay systems so far practically used as tumor markers include those using carcinoembryonic antigens such as carcinoembryonic antigen (CEA), .alpha.-fetoprotein (AFP), Carbohydrate Antigen 125 (CA125); enzymes such as NSE, L-DDC, CK-BB; hormone-related substances such as ACHT, alcohol dehydrogenase (ADH). However, the positive ratio for sera of patients with cancer obtained using the above assay systems is at most 50 to 60%, while frequently patients having cancer provide negative result.
So far, GRP is known as one of tumor markers of small cell lung cancer. GRP is a peptide consisting of 27 amino acids extracted from the stomach of porcine by McDonald in 1978, and has activities to stimulate secretion of gastric acid, various hormones etc. There are three precursor proteins different in their C-terminal structure due to alternative splicing of RNA. Recently, the production of GRP as an autocrine growth factor in small cell lung cancer was found, and it is interested as a tumor marker.
In patients with small cell lung cancer, about 80% of the cases provide increased blood GRP concentration. Moreover, even in the cases of early phase, the blood GRP concentration is in an increased level, and therefore it is promised that a diagnostic of cancer using GRP as a marker is highly effective. However, conventional assay methods for GRP use antibodies to an active peptide, GRP(1-27), and its sensitivity is too low to be practically used. It is supposed that one of main reasons of difficulty to measure a serum GRP concentration using an antibody to GRP(1-27) is instability of GRP(1-27) in the blood.
Holst et al. (J. Clin. Oncol. 7, 1831-1838, (1989)) developed a radioimmunoassay (RIA) system using polyclonal antibodies to a synthetic peptide corresponding to the portion from 42 to 53 positions of C-terminal flanking peptide of GRP precursor, and demonstrated that GRP precursor protein can be a powerful diagnostic marker for small cell lung cancer. However said system was not practical because it provided an insufficient positive ratio due to its sensitivity. Because the antigen protein used was a part of GRP precursor, resulting antibody had low sensitivity and specificity body. Moreover, the low sensitivity of the assay system needed an extraction of the analyte protein from a large amount of a sample, resulting in difficulty in clinical application of the system. GRP precursor protein present in the blood is a macromolecule having a molecular weight of 8,000 to 100,000. Therefor, in an assay system using antibodies to GRP(42-53) which is a part of GRP precursor, sensitivity and specificity are limited.
In normal cells, a protein is produced in the rough-surfaced endoplasmic reticulum, concentrated in the Golgi apparatus resulting in formation of secretory granules in which the protein is packed, and extracellularly secreted through so-called regulated pathway. Since the secretory granules contain proteolytic enzymes, a precursor of the protein can be adequately processed during passing through the regulated pathway. On the other hand, in cancer cells, since the rough-surfaced endoplasmic reticulum is remarkably developed while the number of the secretory granules is small, then when GRP is produced and secreted, GRP precursor is extracellularly secreted through the constitutive pathway from the rough-surfaced endoplasmic reticulum without being affected by action of any proteolytic enzymes, rather than passing through the regulated pathway involving the secretory particles. Therefore, the blood of patients with cancer contains precursor GRP and flanking peptides in addition to an active peptide GRP (1-27).
Compared with GRP(1-27), the active site-free flanking peptide of GRP precursor is expected to be stably present at a high concentration in the blood.